Use of insulin degrading enzyme (IDE) for the treatment of alzheimer&#39;s disease in patients

ABSTRACT

The peptidase insulin degrading enzyme (IDE) has been shown to cleave Aβ peptides. Cleavage of Aβ 1-42  by recombinant IDE abolishes its cell toxicity. Recombinant IDE is expressed on the surface of cells or secreted from cells in plaque forming regions of the brain. Primary hippocampal and cortical cells are treated with Aβ peptides in the presence or absence of IDE and cell toxicity measured. Cells are recombinantly engineered to express IDE on the cell surface or secrete it. The membrane bound and secreted forms of IDE are then expressed in a mammal.

GOVERNMENT INTEREST IN THE INVENTION

[0001] This invention was made with Government support under Grant No. DA 02243 and DA 07062 awarded by the National Institute on Drug Abuse, and Grant No. AG 05893 awarded by the National Institute on Aging. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a method of preventing amyloid plaque formation and/or growth by reacting amyloid peptides with an enzyme that specifically recognizes amyloid peptides, and inactivates them. The present invention also relates to a method of treating Alzheimer's disease by either administering an amyloid peptide degrading enzyme while minimizing or eliminating toxic side effects associated with amyloid peptide byproducts or by increasing the synthesis of the enzyme by administration of pharmacological agents that regulate the expression of the amyloid peptide degrading enzyme.

[0004] 2. Brief Description of the Related Art

[0005] Considerable effort has been expended in identifying the beta and gamma secretases that process the amyloid precursor protein to form the Aβ peptides. The goal of such studies has been to develop specific inhibitors of these enzymes in the hope that such compounds would inhibit the formation of amyloid plaques. The recent report of an aspartyl protease, which appears to be a true beta secretase (R. Vassar et al. (1999) Science 286, 735-741), provides optimism that this approach can soon be tested.

[0006] An alternative strategy is to hydrolyze Aβ peptides before they form amyloid plaques or at least prevent the further development of existing plaques. It may also be possible to remove existing plaques by hydrolyzing any plaque derived Aβ peptide in equilibrium with free Aβ peptide. We test this approach using the zinc metallopeptidase insulin degrading enzyme (IDE, EC. 3.4.22.11), although other peptidases particularly neprilysin can be employed. There are a number of reasons to using IDE for this purpose. First, as noted below, IDE cleaves Aβ₁₋₄₀ and Aβ₁₋₄₂ into what appears to be innocuous products. Second, IDE is a true peptidase; it does not hydrolyze proteins. The enzyme cleaves a limited number of peptides in vitro including insulin and insulin related peptides, β endorphin, and Aβ peptides. Third, cell surface and secreted forms of IDE have been described, and fourth, IDE has been suggested to be one of the physiological Aβ metabolizing enzymes (W. Q. Qui et al. (1998) J. Biol. Chem. 273, 32730-32738).

[0007] Kurichkin and Goto (I. V. Kurochkin and S. Gato (1994) FEBS Lett. 345, 33-37) first reported that insulin degrading enzyme can hydrolyze Aβ₁₋₄₀. This finding was confirmed in two separate studies (W. Q. Qui et al. (1998) J. Biol. Chem. 273, 32730-32738; and J. R. McDermott and A. M. Gibson (1997) Neurochem. Res. 22, 49-56); one of these (W. Q. Qui et al. (1998) J. Biol. Chem. 273, 32730-32738) was a collaboration with this laboratory. Selkoe has proposed that IDE could play a role in determining Aβ peptide levels after their secretion from neuronal and microglial cells (K. Vekrellis et al. (1999) Soc. For Neurosci Abstracts 25, 302). It was suggested that factors that reduce IDE activity, i.e. oxidative damage, can lead to decreased Aβ metabolism and increased amyloid deposits (I. V. Kurochkin and S. Gato (1994) FEBS Lett. 345, 33-37). Although these studies demonstrated that IDE can hydrolyze Aβ₁₋₄₀, they involved the use of either partially purified enzyme preparations such that the products of the reaction could have arisen from secondary cleavages by contaminating peptidases (I. V. Kurochkin and S. Gato (1994) FEBS Lett. 345, 33-37; and J. R. McDermott and A. M. Gibson (1997) Neurochem. Res. 22, 49-56), or the reaction products were not identified (I. V. Kurochkin and S. Gato (1994) FEBS Lett. 345, 33-37). Furthermore, it was not determined whether the products of IDE action on Aβ₁₋₄₀ are neurotoxic or could contribute to amyloid plaque formation, and Aβ₁₋₄₂ was not tested as a substrate. Thus, there is a need in the art for a method of preventing amyloid plaque deposition and methods for treating Alzheimer's disease while minimizing toxic side-effects.

SUMMARY OF THE INVENTION

[0008] The present invention has met the hereinbefore described need.

[0009] It is an object of this invention to provide a method for preventing formation or growth of amyloid fibrils or plaques without causing neurotoxicity, comprising administering an inactivating effective amount of an amyloid peptide inactivating enzyme to a mammal in need thereof. The enzyme may be a peptidase. The enzyme may be an insulin degrading enzyme (IDE), neprilysin or endopeptidase 24.15, endopeptidase 24.16, or similar peptidases.

[0010] It is also an object of the invention to provide a method for preventing formation or growth of amyloid plaque without causing neurotoxicity, comprising:

[0011] a) generating a recombinant viral or plasmid vector comprising a DNA sequence encoding an amyloid peptide inactivating enzyme operatively linked to a promoter;

[0012] b) transfecting in vitro a population of cultured neural cells with said recombinant vector, resulting in a population of transfected neural cells; and

[0013] c) transplanting said transfected neural cells by injection to the brain of a mammalian host, such that expression of said DNA sequence within said brain results in inactivation of said amyloid peptides.

[0014] Another object of the invention to provide a method for preventing formation or growth of amyloid plaque without causing neurotoxicity, comprising:

[0015] a) generating a recombinant viral or plasmid vector comprising a DNA sequence encoding an amyloid peptide inactivating enzyme operatively linked to a promoter; and

[0016] b) injecting said vector to the brain of a mammalian host, such that expression of said DNA sequence within said brain results in inactivation of said amyloid peptides.

[0017] Another object of the invention is to use pharmacological agents to induce the synthesis of endogenous amyloid inactivating enzymes such as insulysin or neprelysin within the brain of affected individuals.

[0018] These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows percent survival of hippocampal cells incubated in media containing Aβ₁₋₄₂ with or without IDE.

[0020]FIG. 2 shows that hippocampal cells treated with IDE are protected against Aβ₁₋₄₂ induced neurotoxic injury.

[0021]FIG. 3 shows that IDE prevents Aβ₁₋₄₀ deposition onto plaques.

[0022]FIG. 4 shows the purification of recombinant rat insulysin, wherein insulysin was purified as described in Examples 4-6 herein and 15 μg aliquots from various stages of purification were analyzed by SDS-PAGE on a 7.5% gel stained with Coomassie Blue. Lane A is Sf9 cell extract. Lane B shows non-bound proteins from the Ni-NTA-agarose column. Lane C shows protein eluted from the Ni-NTA-agarose column with 20 mM imidazole. Lane D shows protein eluted from the Ni-NTA-agarose column with 100 mM imidazole. Lane E shows protein eluted from the Mono-Q column. The position of molecular weight markers (myosin=200 kDa, β-galactosidase=116 kDa, phosphorylase b=97.4 kDa, bovine serum albumin=66 kDa, and ovalbumin=45 kDa) is shown on the left.

[0023]FIG. 5 shows an HPLC profile of products generated from the cleavage of Aβ₁₋₄₀ by insulysin. Varying amounts of recombinant rat insulysin was incubated with 25 μM Aβ₁₋₄₀ for 30 minutes at 37° C. Cleavage products were separated by a 5 to 75% gradient of acetonitrile on a C₄ reverse phase HPLC column. Product peaks are numbered according to their order of elution. The peaks designated Ca and Cb refer to contaminants in the Aβ₁₋₄₀ solution. These are not reacted upon by insulysin as is seen by their invariant peak areas in all the traces. Trace A shows Aβ₁₋₄₀ alone. Trace B shows Aβ₁₋₄₀ incubated with 50 ng insulysin. Trace C shows Aβ₁₋₄₀ incubated with 250 ng insulysin. Trace D shows Aβ₁₋₄₀ incubated with 500 ng insulysin. The HPLC scans are skewed ˜2 min. to the left to permit overlapping peaks to be viewed. The time scale refers to trace A.

[0024]FIG. 6 shows positions of cleavage within the Aβ₁₋₄₀ and Aβ₁₋₄₂ sequences. The primary cleavage sites are noted with the thick arrows.

[0025]FIGS. 7A and 7B show the effect of insulysin on the neurotoxic effects of Aβ peptides. Purified insulysin was added with Aβ₁₋₄₀ (30 μM) or Aβ₁₋₄₂ (25 μM) to primary cortical neurons, and incubation continued for an additional 48 hrs. The neurotoxic effect of the Aβ peptides was determined as described in Example 9 herein. The insulysin and heat inactivated insulysin controls utilized 5000 ng of enzyme. FIG. 7A shows the effect of incubation with insulysin on the neurotoxic effects of Aβ₁₋₄₀. FIG. 7B shows the effect of incubation with insulysin on the neurotoxic effects of Aβ₁₋₄₂. * designates p=<0.01 relative to the Aβ treated sample as determined by ANOVA.

[0026]FIG. 8 shows that insulysin protects against Aβ₁₋₄₀ mediated neurotoxicity. Rat cortical neurons were treated as described in FIG. 7 in the presence or absence of 50 ng insulysin. Cells were stained with Hoechst 33258 (panels A-D) or with the Aβ antibody 10D5 (panels E-H). Hoffman modulation contrast micrographs are shown in panels I-L. Panels A, E, and I show untreated neurons. Panels B, F and J show neurons with 50 ng insulysin added. Panels C, G and K show neurons treated with 30 μM Aβ₁₋₄₀ Panels D, H and L show neurons treated with 50 ng insulysin and 30 μM Aβ₁₋₄₀

[0027]FIGS. 9A and 9B show that insulysin inhibits the deposition of Aβ₁₋₄₀ onto synthetic amyloid plaques. FIG. 9A shows the effect of incubation with insulysin on the deposition of Aβ₁₋₄₀. Aβ₁₋₄₀ (0.1 nM) was mixed with the indicated amount of purified insulysin and then added to synthaloid in 96 well plates. Deposition was permitted to occur over a 4 hr time period. FIG. 9B shows the effect of preincubation with insulysin on the deposition of Aβ₁₋₄₀. Aβ₁₋₄₀ (1 nM) was preincubated for 60 minutes the indicated amount of purified insulysin. The incubation mixtures were then added to synthaloid in 96 well plates and deposition was permitted to occur over a 4 hr time period. * indicates P=<0.01 as determined by ANOVA.

DETAILED DESCRIPTION OF THE INVENTION

[0028] As used herein, the term “patient” includes members of the animal kingdom including but not limited to human beings.

[0029] As used herein, the term “mammalian host” includes members of the animal kingdom including but not limited to human beings.

[0030] As used herein, the term “brain tissue” refers to tissue that comprises neural tissue, including hippocampal and cortical tissue.

[0031] As used herein, “amyloid peptide inactivating enzyme” encompasses a group of functionally or structurally related proteins that bind specifically to amyloid peptides, and prevent the peptides from depositing as plaques or fibrils. Preferably, toxic side-effects are minimized. By “inactivating” it is meant that the enzyme may functionally prevent amyloid peptides from forming plaques. Preferably, “inactivating” refers to degradation of the amyloid peptide. More preferably, the enzyme is a peptidase. Most preferably, the enzyme is insulin degrading enzyme or neprilysin (endopeptidase 24.11), although other possiblilities include endopeptidase 24.15 (EC. 3.4.24.15) or endopeptidase 24.26 (EC. 3.4.24.16), or similar peptidases. The invention is not limited to these enzymes.

[0032] As used herein, “amyloid peptide” includes beta or gamma amyloid peptides. Preferably, the peptide is amyloid beta peptide. More preferably, the beta peptide is Aβ₁₋₄₀ or Aβ₁₋₄₂.

[0033] As used herein, “selectable marker” includes a gene product that is expressed by a cell that stably maintains the introduced DNA, and causes the cell to express an altered phenotype such as morphological transformation, or an enzymatic activity. Isolation of cells that express a transfected gene is achieved by introduction into the same cells a second gene that encodes a selectable marker, such as one having an enzymatic activity that confers resistance to an antibiotic or other drug. Examples of selectable markers include, but are not limited to, thymidine kinase, dihydrofolate reductase, aminoglycoside phosphotransferase, which confers resistance to aminoglycoside antibiotics such as kanamycin, neomycin and geneticin, hygromycin B phosphotransferase, xanthine-guanine phosphoribosyl transferase, CAD (a single protein that possesses the first three enzymatic activities of de novo uridine biosynthesis—carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase), adenosine deaminase, and asparagine synthetase (Sambrook et al. Molecular Cloning, Chapter 16. 1989), incorporated herein by reference in its entirety.

[0034] As used herein, a “promoter” can be any sequence of DNA that is active, and controls transcription in an eucaryotic cell. The promoter may be active in either or both eucaryotic and procaryotic cells. Preferably, the promoter is active in mammalian cells. The promoter may be constitutively expressed or inducible.

[0035] As used herein, the term “DC-chol” means a cationic liposome containing cationic cholesterol derivatives. The “DC-chol” molecule includes a tertiary amino group, a medium length spacer arm (two atoms) and a carbamoyl linker bond (Gao et al., Biochem. Biophys. Res, Commun., 179:280-285, 1991).

[0036] As used herein, “SF-chol” is defined as a type of cationic liposome.

[0037] As used herein, the term “biologically active” used in relation to liposomes denotes the ability to introduce functional DNA and/or proteins into the target cell.

[0038] As used herein, the term “biologically active” in reference to a nucleic acid, protein, protein fragment or derivative thereof is defined as an ability of the nucleic acid or amino acid sequence to mimic a known biological function elicited by the wild type form of the nucleic acid or protein.

[0039] As used herein, the term “maintenance”, when used in the context of liposome delivery, denotes the ability of the introduced DNA to remain present in the cell. When used in other contexts, it means the ability of targeted DNA to remain present in the targeted cell or tissue so as to impart a therapeutic effect.

[0040] The present invention discloses ex vivo and in vivo techniques for delivery of a DNA sequence of interest to the brain tissue cells of the mammalian host. The ex vivo technique involves culture of target brain tissue cells, in vitro transfection of the DNA sequence, DNA vector or other delivery vehicle of interest into the brain tissue cells, followed by transplantation of the modified brain tissue cells to the target joint of the mammalian host, so as to effect in vivo expression of the gene product of interest.

[0041] As an alternative to the in vitro manipulation of brain tissue cells, the gene encoding the product of interest is introduced into liposomes and injected directly into the area of the joint, where the liposomes fuse with the brain tissue cells, resulting in an in vivo gene expression of the amyloid peptide inhibiting enzyme.

[0042] As an additional alternative to the in vitro manipulation of brain tissue cells, the gene encoding the product of interest is introduced into the area of the brain as naked DNA. The naked DNA enters the brain tissue cell, resulting in an in vivo gene expression of the amyloid peptide inhibiting enzyme.

[0043] Still another alternative is to use pharmacological agents to induce synthesis of the endogenous gene encoding the amyloid peptide inhibiting enzyme. Such a pharmacological substance may be a compound that “up regulates” or enhances the expression of the amyloid peptide inhibiting enzyme. The pharmacological agent may bind to the regulatory region of the gene encoding the enzyme and thus activate its gene expression. Thus, the compound may be a transcriptional activator of the gene encoding the enzyme. Or, the compound may have a regulatory effect post transcriptionally in, for example, stabilizing the amyloid peptide inhibiting enzyme structure.

[0044] The pharmacological agent may be placed in pharmaceutically acceptable excipient or carrier and administered to a person or individual in need thereof. Depending on the specific clinical status of the disease, administration can be made via any accepted systemic delivery system, for example, via oral route or parenteral route such as intravenous, intramuscular, subcutaneous or percutaneous route, or vaginal, ocular or nasal route, in solid, semi-solid or liquid dosage forms, such as for example, tablets, suppositories, pills, capsules, powders, solutions, suspensions, cream, gel, implant, patch, pessary, aerosols, collyrium, emulsions or the like, preferably in unit dosage forms suitable for easy administration of fixed dosages. The pharmaceutical compositions will include a conventional carrier or vehicle and the pharmacological compound and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, and so on.

[0045] If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and so on.

[0046] The compounds of this invention are generally administered as a pharmaceutical composition which comprises a pharmaceutical vehicle in combination with the pharmacological compound. The amount of the drug in a formulation can vary within the full range employed by those skilled in the art, e.g., from about 0.01 weight percent (wt %) to about 99.99 wt % of the drug based on the total formulation and about 0.01 wt % to 99.99 wt % excipient.

[0047] The preferred mode of administration, for the conditions mentioned above, is oral administration using a convenient daily dosage regimen which can be adjusted according to the degree of the complaint. For said oral administration, a pharmaceutically acceptable, non-toxic composition is formed by the incorporation of the selected pharmacological compound in any of the currently used excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. Such compositions take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like. Such compositions may contain between 0.01 wt % and 99.99 wt % of the active compound according to this invention.

[0048] Preferably the compositions will have the form of a sugar coated pill or tablet and thus they will contain, along with the active ingredient, a diluent such as lactose, sucrose, dicalcium phosphate, and the like; a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such as starch, polyvinylpyriolidone, acacia gum, gelatin, cellulose and derivatives thereof, and the like.

[0049] It is understood that by “pharmaceutical composition”, it is meant that the pharmacological compound is formulated into a substance that is to be administered purposefully for inactivating the amyloid protein. The mode of action is believed to be by cleavage of the amyloid inactivating protein. However, it is understood that the pharmacological compound per se will not have a toxic effect, and by “pharmaceutical composition”, it excludes those compositions that are used to administer to individuals as test compounds for a purpose other than as an inducer of inactivation of the amyloid protein.

[0050] It will be understood by the artisan of ordinary skill that the preferred source of cells for treating a human patient is the patient's own tissue cells, such as autologous brain hippocampal or cortical cells or even fibroblasts.

[0051] A further embodiment of the present invention includes employing as the gene a gene capable of encoding an amyloid peptide inactivating enzyme or a biologically active derivative or fragment thereof, and employing as the DNA plasmid vector any DNA plasmid vector known to one of ordinary skill in the art capable of stable maintenance within the targeted cell or tissue upon delivery, regardless of the method of delivery utilized.

[0052] One such method is the direct delivery of the DNA vector molecule, whether it be a viral or plasmid DNA vector molecule, to the target cell or tissue. This method also includes employing as the gene a gene capable of encoding an amyloid peptide inactivating enzyme or biologically active derivative or fragment thereof.

[0053] Another embodiment of this invention provides a method for introducing at least one gene encoding a product into at least one cell of a brain tissue for use in treating the mammalian host. This method includes employing non-viral means for introducing the gene coding for the product into the brain tissue cell. More specifically, this method includes a liposome encapsulation, calcium phosphate coprecipitation, electroporation, or DEAE-dextran mediation, and includes employing as the gene a gene capable of encoding an amyloid peptide inactivating enzyme or biologically active derivative or fragment thereof, and a selectable marker, or biologically active derivative or fragment thereof.

[0054] Another embodiment of this invention provides an additional method for introducing at least one gene encoding a product into at least one cell of a brain tissue for use in treating the mammalian host. This additional method includes employing the biologic means of utilizing a virus to deliver the DNA vector molecule to the target cell or tissue. Preferably, the virus is a pseudovirus, the genome having been altered such that the pseudovirus is capable only of delivery and stable maintenance within the target cell, but not retaining an ability to replicate within the target cell or tissue. The altered viral genome is further manipulated by recombinant DNA techniques such that the viral genome acts as a DNA vector molecule which contains the heterologous gene of interest to be expressed within the target cell or tissue.

[0055] A preferred method of the present invention involves direct in vivo delivery of an amyloid peptide inhibiting enzyme gene to the brain tissue of a mammalian host through use of either an adenovirus vector, adeno-associated virus (AAV) vector, lentivirus, or herpes-simplex virus (HSV) vector. In other words, a DNA sequence of interest encoding a functional amyloid peptide inhibiting enzyme or enzyme fragment is subcloned into the respective viral vector. The amyloid peptide inhibiting enzyme gene containing viral vector is then grown to adequate titer and directed into the brain, preferably by cortical or hippocampal injection.

[0056] Direct brain tissue injection of a DNA molecule containing the gene of interest results in transfection of the recipient brain tissue cells and hence bypasses the requirement of removal, in vitro culturing, transfection, selection, as well as transplanting the DNA vector containing-fibroblast to promote stable expression of the heterologous gene of interest.

[0057] In a preferred embodiment, using homogeneous recombinant IDE we have found that the enzyme cleaves both Aβ₁₋₄₀ and Aβ₁₋₄₂ at the His¹³-His¹⁴, His¹⁴-Gln¹⁵, and Phe¹⁹-Phe²⁰ bonds. More importantly, using a cell toxicity assay, we show that the peptides derived from the IDE catalyzed hydrolysis of Aβ₁₋₄₂ are not neurotoxic to hippocampal neurons, presumably because they do not aggregate. Thus IDE is neuroprotective.

[0058] In the first aim of this application we characterize the ability of IDE to act as a neuroprotective agent and determine if IDE can prevent β-amyloid deposition. The second objective is to engineer IDE so as to either have it secreted from cells or to have it expressed on the plasma membrane. Neprilysin is normally expressed on the cell surface and can be engineered to be secreted. Viral vectors are used to express IDE in hippocampal and cortical neurons to show that these cells become resistant to the neurotoxic effects of Aβ peptides. The third objective is to express these constructs in the R1.40-Homo-G9 Hemi transgenic mouse model of Alzheimer's disease (B. T. Lamb et al. (1999) Nat. Neurosci. 2, 695-6697). We test this gene therapy approach to see if IDE and neprilysin (NEP) prevents plaque formation and promotes the dissolution of preformed amyloid plaques. The application is also directed to using gene therapy to treat Alzheimer's patients.

[0059] The balance between the anabolic and catabolic pathways in the metabolism of the Aβ peptides is a delicate one. Although considerable effort has focused on the generation of the Aβ peptides, until recently considerably less emphasis has been placed on the clearance of these peptides. Removal of extracellular Aβ peptide appears to proceed through two general mechanisms; cellular internalization and extracellular degradation by neuropeptidases. Apparently neither of these mechanisms is adequate in Alzheimer's disease. Interest in the mechanism of cellular internalization stems from the apparent involvement of apolipoprotein E and α-2-macroglobulin in this process (Narita et al. (1997) J. Neurochem 69:1904-1911; Hughes et al. (1998) Proc Natl Acad Sci U S A 95:3275-3280; Kang et al. (1997) Neurology 49:56-61; Blacker et al. (1998) Nat Genet 19:357-360).

[0060] We have expressed a His₆-tagged version of rat insulin degrading enzyme (IDE) in Sf9 cells using the baculovirus expression system. The purified recombinant IDE is fully active either with the His₆-tag removed by cleavage at a TEV protease site or with the His₆-tag intact. This purified recombinant IDE has been used to analyze the cleavage of Aβ₁₋₄₀ and Aβ₁₋₄₂ using MALDI-TOF and EMSI mass spectrometry to identify cleavage products. These experiments showed cleavage of both Aβ₁₋₄₀ and Aβ₁₋₄₂ at the His¹³-His¹⁴, His¹⁴-Gln¹⁵, and Phe¹⁹-Phe²⁰ bonds.

[0061] Cleavage of both Aβ₁₋₄₀ and Aβ₁₋₄₂ by the recombinant enzyme was shown to initially occur at the His¹³-His¹⁴, His¹⁴-Gln¹⁵, and Phe¹⁹-Phe²⁰ bonds. This was followed by a slower cleavage at the Lys²⁸-Gly²⁹, Val¹⁸-Phe¹⁹ and Phe²⁰-Ala²¹ positions. None of the products appeared to be further metabolized by insulysin. Using a rat cortical cell system, the action of insulysin on Aβ₁₋₄₀ and Aβ₁₋₄₂ was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown to prevent the deposition of Aβ₁₋₄₀ onto a synthetic amyloid. Taken together these results suggest that the use of insulysin to hydrolyze Aβ peptides represents an alternative gene therapeutic approach to the treatment of Alzheimer's disease.

[0062] The cleavage products observed with insulysin indicate distinct cleavage events and not products derived from secondary cleavage of an initial product. That is, no fragment was observed lacking an intact N-terminus, the C-terminal fragment corresponding to each N-terminal fragment was seen in all but one case, and products increased with an increasing concentration of insulysin.

[0063] Neuronal cell cultures are susceptible to the toxic effects mediated by Aβ₁₋₄₀ and Aβ₁₋₄₂. We have used this neuronal cell culture system to establish that the products of the insulysin dependent cleavage of Aβ₁₋₄₀ and Aβ₁₋₄₂ produces products that are not in themselves neurotoxic. This is an important point if one were to consider the use of insulysin in the treatment of Alzheimer's disease.

[0064] Related to cellular toxicity, Aβ peptides are able to deposit onto an existing matrix of peptides in what is thought to lead to an increase in the size of senile plaques and consequently to the progression of Alzheimer's disease. In a model system, Esler et.al. (Esler et al. (1997) Nat Biotech 15:268-263) have shown that the deposition of Aβ₁₋₄₀ onto a preformed synthaloid matrix mimics the in vivo deposition of Aβ peptides onto the brain cortex. Using this model, we have shown that insulysin cleavage of Aβ₁₋₄₀ prevents the deposition of the Aβ peptides onto the synthaloid. This suggests that insulysin may be able to prevent the formation and growth of senile plaques in Alzheimer's disease patients.

[0065] In summary we have established that the insulysin dependent cleavage of the Aβ peptides leads to the loss of both their neurotoxic properties as well as their ability to contribute to plaque formation and growth. The use of insulysin and other peptidases to degrade extracellular Aβ peptides represents a new approach toward the treatment of Alzheimer's disease.

[0066] An objective of this patent application is to further describe and characterize the ability of IDE to act as a neuroprotective agent. We measure the ability of IDE to protect cultured hippocampal and cortical cells from the toxic effects of Aβ₁₋₄₀ and Aβ₁₋₄₂. For these experiments primary hippocampal and cortical cells are obtained from 18 day rat embryos as described by Mattson et al. (M. P. Mattson et al. (1995) J. Neurochem. 65, 1740-1751) and initially cultured for seven days in Eagles MEM supplemented with fetal bovine serum, KCl, pyruvate, and gentamicin as described by Lovell et al (M. A. Lovell et al. (1999) Brain Res. 823, 88-95). Prior to use, the cells are transferred to Locke's media and dispersed in 96 well plates at a density of ˜10⁵ cells/well. Cells are then treated in triplicate with varying concentrations of Aβ₁₋₄₀ and Aβ₁₋₄₂ (1 to 25 μM) for up to 48 hrs. Toxicity of the Aβ peptides are quantitated at various times by measuring MTT oxidation and LDH release (C. Behl et al. (1994) Cell 77, 817-827) using assay kits from Promega Corp. (Promega CytoTox96® Non-Radioactive Cytotoxicity Assay Kit). Another set of cultures will have added to them 5 to 500 ng of purified IDE previously dialyzed into Locke's media and filter sterilized. We have previously established that IDE is fully active in Locke's media under cell culture conditions for several days. As a control, IDE inactivated by removal of its zinc cofactor by treatment with o-phenanthroline, and then dialyzed to remove the o-phenanthroline, are used. Another control will have IDE added to the cultured cells in the absence of Aβ peptides.

[0067] A variation of this protocol is to pre-aggregate the Aβ peptides prior to their addition to the cultured cells. For aggregation, Aβ peptide is incubated in Locke's media and the formation of aggregates followed by measuring an increase in turbidity at 400 nm. The Aβ peptide is allowed to maximally aggregate before use. The aggregated Aβ peptide is then added to the primary cultures as noted above in the presence or absence of IDE, and toxicity determined as indicated above. Under this experimental condition IDE will be protective if it can hydrolyze Aβ in the aggregated state or if aggregation is rapidly reversible and the free Aβ can be broken down by IDE.

[0068] The next set of experiments utilizes a “more physiological” Aβ deposition assay in which physiological concentrations (˜10⁻⁹ M) of ¹²⁵I-Aβ are deposited onto a preformed synthetic amyloid (synthaloid) in a 96 well plate (W. P. Esler et al. (1999) Meth. In Enz. 309, 350-374). The assay is readily quantitated by measuring the ¹²⁵I deposited onto the plate. The 96 well plates containing synthaloid are available commercially from QCB/BioSource and ¹²⁵I-Aβ is available from Amersham. This assay is used to determine if IDE can prevent Aβ₁₋₄₀ and Aβ₁₋₄₂ deposition. Varying amounts of IDE are added to incubation mixtures containing ¹²⁵I-Aβ (˜100 pM) in Tris buffer and the rate of radiolabeled Aβ deposition in the presence and absence of IDE are compared. Ortho-phenanthroline treated IDE is used as a control. Experiments suggest that IDE hydrolysis products of Aβ₁₋₄₀ do not deposit onto the synthaloid.

[0069] A variation of this assay is used to see if IDE can release newly deposited Aβ. In this assay ¹²⁵I-Aβ is deposited onto preformed synthaloid for 2-4 hrs, free Aβ is washed away, and then buffer is added with or without IDE. The supernatant is counted at various times to see if the newly deposited Aβ is solubilized. This assay is also used to see if IDE can “dissolve” preformed amyloid plaques. In these experiments ¹²⁵I-Aβ is used during the preparation of the synthaloid which will permit it to become an integral part of the synthetic amyloid aggregate. IDE or control inactive IDE is added to the pre-formed ¹²⁵I-Aβ synthaloid and incubated for varying lengths of time. The amount of ¹²⁵I released into the media is then measured. As noted above ¹²⁵I release occurs if IDE can act directly on the Aβ fibrils or if there is a dynamic equilibrium between free Aβ and Aβ in the plaque.

[0070] Taken together these experiments demonstrate the usefulness of IDE to protect against both the neurotoxicity of Aβ and the ability of Aβ to be deposited onto amyloid plaques.

[0071] The second objective is to engineer the IDE molecule so as to have it either expressed as an extracellular plasma membrane protein or be secreted. NEP will be engineered to be secreted. Such forms of IDE and NEP are introduced into primary hippocampal cells through a viral vector and should make these cells resistant to the neurotoxic effects of Aβ peptides. Previous studies (K. Vekrellis et al. (1999) Soc. For Neurosci Abstracts 25, 302; and K. A. Seta and R. A. Roth (1997) Biochem. Biophys. Res. Commun. 231, 167-171) have shown that a small fraction of IDE can be expressed on the cell surface and that IDE can be secreted into the media. NEP is normally found on the cell surface. Thus IDE can be transported to the cell surface and fold properly, however this appears to be an inefficient process as most of the IDE is found within the cell. Two domains from the β subunit of the peptidase meprin (G. Johnson, G. and L. B. Hersh, L. B. (1992) J. Biol. Chem. 267, 13505-13512) are used to place IDE on the cell surface. The C-terminal region of the rat meprin β subunit has been shown to anchor the protein to the plasma membrane while the N-terminal region of rat meprin β has a secretion signal (G. Johnson, G. and L. B. Hersh, L. B. (1994) J. Biol. Chem. 269, 7682-7688). The rat meprin β subunit cDNA was originally cloned in this laboratory and thus we have experience working with both the protein and its cDNA. The cDNA is used as a template for PCR to obtain the C and N-terminal coding sequences and ligate them to the rat IDE cDNA. The fusion at the C-terminal region is such that the SKL peroxisomal targeting signal found at the C-terminus of IDE is removed. The construct is assembled initially in pBluescript and then transferred to the adenovirus expression vector system of He et al. (T -C He et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2509-2514) This system permits the generation of a recombinant adenoviral plasmid in E. coli, and the use of this plasmid to obtain virus from mammalian cells (i.e. 911E4 cells) without the need for plaque purification. It greatly facilitates the generation of recombinant adenovirus constructs.

[0072] To obtain a secreted form of IDE we either simply leave off the C-terminal domain of the rat meprin β subunit or substitute the C-terminal domain of the rat meprin α subunit for the C-terminal domain of the rat meprin β subunit. We have shown that the C-terminal domain of the rat meprin α subunit, although very similar in sequence to the β subunit, is efficiently cleaved and secreted from cells (G. Johnson, G. and L. B. Hersh, L. B. (1994) J. Biol. Chem. 269, 7682-7688).

[0073] The virus constructs containing the modified IDE or NEP forms are initially expressed in CHO cells to test targeting to the cell surface or secretion. This is accomplished in two ways. Plasma membrane expression is determined using cell surface biotinylation with biotinamidocaproic acid 3-sulfo-N-hydroxysuccimimide a cell impermeable labeling reagent, which has been shown to label plasma membrane IDE (K. A. Seta and R. A. Roth (1997) Biochem. Biophys. Res. Commun. 231, 167-171). IDE expressed as an intracellular protein is used as a control. Secondly, we demonstrate that the surface expressed IDE and NEP is enzymatically active by incubating cells expressing IDE or NEP on the surface with β-endorphin, a good IDE substrate (A. Safavi et al. (1996) Biochemistry 35, 14318-14325), and showing that the extracellular, but not the intracellular form of IDE, can degrade β-endorphin. HPLC is used to follow β-endorphin hydrolysis (A. Safavi et al. (1996) Biochemistry 35, 14318-14325). We have previously used this protocol to study the degradation β-endorphin by intact macrophages (B. Sarada, D. Thiele et al. (1997) J. Leukocyte Biol. 62, 753-760). Although insulin is the most widely used substrate for the enzyme, the possibility that it would be internalized through insulin receptors and degraded intracellularly precludes its use.

[0074] Western blot analysis of conditioned media as well as the measurement of β-endorphin hydrolysis by conditioned media from cells expressing the secreted form of IDE or NEP is used to measure secretion of the enzyme. A control includes cells expressing intracellular IDE or membrane associated NEP.

[0075] Once we demonstrate that IDE and NEP are expressed on the plasma membrane or secreted we express these IDE forms in primary hippocampal and cortical cells through the adenovirus vector. Intracellularly expressed IDE is used as a control. The IDE and NEP expressing hippocampal and cortical cells are tested for their sensitivity to the toxic effects of Aβ₁₋₄₀ and Aβ₁₋₄₂ as described above. We compare the concentration dependence and time dependence of Aβ₁₋₄₀ and Aβ₁₋₄₂ induced cell toxicity as described above. We adapt the Aβ deposition assay such that these modified cells are added to the 96 well plates during the assay. We then determine the effectiveness of secreted or cell surface expressed IDE and NEP in preventing Aβ deposition. These experiments permit us to assess the use of IDE and NEP to prevent amyloid fibrils and plaques in vitro.

[0076] After analyzing the in vitro data, we express cell surface or secreted IDE and NEP in a transgenic mouse model of Alzheimer's disease. We use the R1.40-HomoxG9-Hemi mouse recently described by Lamb et al (B. T. Lamb et al. (1999) Nat. Neurosci. 2, 695-697), which is a cross of a G9 mouse, which expresses the entire human presenilin-1 gene containing the H163R mutation with an R1.40 mouse, which contains the entire human APP gene. These mice develop fibrillar Aβ deposits in the frontal cortex and hippocampus within seven months (B. T. Lamb et al. (1999) Nat. Neurosci. 2, 695-697). Thus, the use of this mouse model greatly reduces the time needed to assess the results of these studies.

[0077] At ages from 1-6 months we introduce into the right frontal cortex or right hippocampus our adenovirus or lentivirus constructs expressing secreted or cell surface forms of IDE and NEP. Adenovirus injections are made using a Hamilton syringe with a 33-gauge needle mounted on a Kopf stereotaxic device. Varying amounts of virus are initially tested in order to produce maximal cell infection and expression of the transgene. Mice at 2, 4, and 6 months are sacrificed to test for both the efficiency of infection (i.e. number of cells expressing the IDE or NEP transgene) and the length of continued expression of the transgene using IDE and meprin immunohistochemistry or NEP immunohistochemistry. To increase the expression time of the transgene and decrease cellular immunity we use an adenovirus (Ad5) containing a temperature sensitive DNA binding protein as well as injecting monoclonal Aβs against CD4 and CD45 to immunosuppress the animals (M. I. Romero and G. M. Smith (1998) Gene Therapy 5, 1612-1621). This regimen has been shown to effectively increase expression of the transgene as well as permit multiple injections of adenovirus (M. I. Romero and G. M. Smith (1998) Gene Therapy 5, 1612-1621). The temperature sensitive adenovirus has been found to express transgenes in mice for 3-4 months (M. I. Romero and G. M. Smith (1998) Gene Therapy 5, 1612-1621). Lentivirus can be used directly. An alternative approach is to use a cellular promoter, i.e. the β-actin promoter, in our virus construct since it has been shown that cellular promoters express longer than the standard viral promoters commonly used with virus vectors (G. M. Smith and M. I. Romero (1999) J. Neurosci. Res. 55, 147-157).

[0078] Once optimal amounts of virus and the number of times it needs to be introduced to maintain cells expressing IDE on the surface or secreted are determined, we examine the effect of these IDE and NEP forms in preventing fibrillar Aβ deposits in the R1.40-HomoxG9-Hemi mouse. We stain treated and control mice (virus with intracellular form of IDE) on the injected side and the contralateral side with thioflavin S and silver using standard histochemical methods (D. R. Borchelt et al. (1997) Neuron 19, 939-945). A quantitative estimate of the effectiveness of IDE and NEP in preventing or reducing amyloid deposits is obtained by immunoctyochemical measurement of β-amyloid load as described by Geddes (T. L. Tekirian et al. (1998) J. Neuropath. Exp. Neurol. 57, 76-94). It is expected that fewer fibrillar Aβ deposits are seen on the injected side in treated mice compared to control mice.

[0079] Next, if the IDE and NEP expressed on the cell surface or secreted can prevent Aβ deposition, we use the same paradigm to see if either of these IDE and NEP forms affect preformed Aβ deposits. In this case the R1.40-HomoxG9-Hemi mice are treated with the IDE and NEP virus constructs at time periods of seven to nine months, a time at which the Aβ deposits will have already formed (B. T. Lamb et al. (1999) Nat. Neurosci. 2, 695-6697). We compare the treated and contralateral side as well as treated and untreated mice to see if the introduced IDE and NEP has decreased the number of Aβ deposits.

[0080] Taken together these experiments provide an indication as to IDE's and NEP's use in preventing Aβ deposition in Alzheimer's patients.

[0081] Other possibilities include endopeptidase 24.15 (E.C. 3.4.24.15) or endopeptidase 24.26 (E.C. 3.4.24.16), or similar peptidases.

[0082] The following examples are offered by way of illustration of the present invention, and not by way of limitation.

EXAMPLES Example 1

[0083] In order to determine whether the IDE cleavage of Aβ peptides produces products which in themselves are neurotoxic, we conducted experiments using cultured primary hippocampal cells. In this experiment we preincubated Aβ₁₋₄₂ with IDE and compared the effect of the IDE treated Aβ₁₋₄₂ to the intact peptide. An inactivated form of IDE was used as a control. In a second paradigm we added IDE directly to hippocampal cell cultures at the same time in which these cells were treated with Aβ₁₋₄₂. In both types of experiments, treatment with IDE prevented Aβ₁₋₄₂ induced cell death.

[0084] Referring to FIG. 1, A represents control hippocampal cells incubated in media for 24 hrs. B is the same as A treated with 10 μM Aβ₁₋₄₂ (initially monomeric). C is the same as A treated with 10 μM Aβ₁₋₄₂ (initially monomeric)+400 ng of insulin degrading enzyme. D is the same as A treated with 10 μM Aβ₁₋₄₂ (initially monomeric)+400 ng of inactive insulin degrading enzyme. Viable cells were detected by microscopy.

Example 2

[0085] It has been previously established that a culture of rat brain hippocampal neurons is a good model for studying the neurotoxicity of amyloid peptides towards neurons in brains of patients with Alzheimer's disease. The addition of amyloid beta peptide (Aβ₁₋₄₂) to the hippocampal cell cultures has been shown to be sufficiently toxic and is thought to accurately reflect the action of Aβ₁₋₄₂ in patients' brains. The object of this experiment was to see if insulin degrading enzyme (IDE) could break down Aβ₁₋₄₂ into fragments that are no longer neurotoxic. A setup was used where rat brain cells were treated with 25 μM Aβ₁₋₄₂ in the absence and presence of IDE. The results show that the cells treated with Aβ₁₋₄₂ and IDE were protected from oxidative damage.

[0086] Methods

[0087] Rat hippocampal cells were taken in culture dishes and treated with 25 μM Aβ₁₋₄₂ in the presence and absence of IDE for up to 12 hours. Neuronal survival was estimated as a function of time. Untreated hippocampal cells were relatively unaffected after 12 hours while cells treated with 25 μM Aβ₁₋₄₂ decreased to 20% of the initial number after 12 hours. When IDE was added with 25 μM Aβ₁₋₄₂ to the cells, survival was close to that seen in the control untreated cells. Heat killed IDE was used as a control to show that the neuroprotection seen with IDE required enzymatically active IDE.

Example 3

[0088] A protocol was used where amyloid beta 1-40 (Aβ₁₋₄₀) is initially deposited onto a 96 well microtiter plate. Radioactive (¹²⁵I labeled) Aβ₁₋₄₀ is then added to the wells of this plate where it further adds to the Aβ₁₋₄₀ deposited. This mimics the deposition of Aβ₁₋₄₀ seen in the brains of Alzheimer patients.

[0089] The object of this experiment was to see if insulin degrading enzyme (IDE) could break down Aβ₁₋₄₀ into fragments that are no longer deposited on the amyloid plaques. This demonstrates that IDE could prevent the continued formation of amyloid deposits in Alzheimer's disease.

[0090] Methods

[0091] 96 well plates were pre-coated with Aβ₁₋₄₀. In the control, 100 pM of ¹²⁵I-Aβ₁₋₄₀ was deposited onto the pre-deposited Aβ₁₋₄₀ plaque for three hours (lane 1). IDE was added at concentrations of 500 ng, 50 ng and 5 ng to the wells along with ¹²⁵I-Aβ₁₋₄₀ for three hours is (lanes 2 to 4). 50% inhibition of deposition of ¹²⁵I-Aβ₁₋₄₀ was seen with 50 ng of IDE.

Example 4 Materials

[0092] Aβ₁₋₄₀ and Aβ₁₋₄₂ were obtained from Bachem (Torrance, Calif.). Solutions were prepared by dissolving the peptide in dimethylsulfoxide (DMSO) to give a stock concentration of 200 μM. The peptide stock was lyophilized and stored at −80° C. until use. The aggregation state of Aβ peptide stock solutions was checked by electron microscopy (Ray et al. (2000) Brain Res 853:344-351) and found to be predominantly, if not exclusively, monomeric. For the in vitro reactions with insulysin, a final concentration of 25 μM Aβ₁₋₄₀ was obtained after bringing the lyophilized peptide into solution with double distilled water. For cytotoxicity studies Aβ₁₋₄₀ and Aβ₁₋₄₂ peptides were dissolved in sterile N2 medium (Life Technologies, Rockville, Md.). Human β-endorphin₁₋₃₁, obtained from the National Institute on Drug Abuse drug supply system, was dissolved in water to give a stock solution of 300 μM. Trifluoroacetic acid (Sigma Biochemicals, St. Louis, Mo.) was diluted into water to produce a 5% working solution.

Example 5 Expression and Purification of Recombinant Insulysin

[0093] A rat insulysin cDNA, (pECE-IDE) was subcloned into the baculovirus derived vector pFASTBAC (GIBCO BRL, Rockville, Md.) through BamH I and Xho I restriction sites such that a His₆-affinity tag was attached to the N-terminus of the protein. Generation of recombinant virus and expression of the recombinant protein in Sf9 cells was carried out according to the manufacturer's directions. For the purification of recombinant insulysin, a 1/10 (wt/vol.) suspension derived from a 50 ml culture of viral infected Sf9 cells was prepared in 100 mM potassium phosphate buffer, pH 7.2, containing 1 mM dithiothreitol (K—PO₄/DTE buffer). The suspension was sonicated 10 times, each burst for one second, using a Branson sonifier (setting 3 at 30%) and then centrifuged at 75,000 g for 30 minutes to pellet cell debris and membranes. The supernatant containing recombinant rat insulysin was loaded onto a 0.5-ml nickel-NTA column (Qiagen, Valencia, Calif.) that had been equilibrated with the K—PO₄/DTE buffer. After extensive washing of the column with starting buffer, and then with 20 mM Imidazole-HCl, pH 7.2, the enzyme was eluted with 0.1 M Imidazole-HCl, pH 7.2. The enzyme was further purified over a 1 ml Mono-Q anion exchange column (Pharmacia Biotech, Piscataway, N.J.) in 20 mM phosphate buffer pH 7.2. A linear salt gradient of 0 to 0.6 M KCl, equivalent to 60 column volumes, was applied to the column with the enzyme eluted at 0.28 M KCl. SDS-PAGE of the insulysin was conducted on a 7.5% gel.

Example 6 Insulysin Activity Determination

[0094] Insulysin activity was assayed by measuring the disappearance of β-endorphin by isocratic reverse phase HPLC (Safavi et al. (1996) Biochemistry 1996 35:14318-14325). A 100 μl reaction mixture containing 40 mM potassium phosphate buffer, pH 7.2, 30 μM β-endorphin, and enzyme was incubated for 15 minutes at 37° C. The reaction was stopped by the addition of 10 μl of 5% trifluoroacetic acid to give a final concentration of 0.5%. The reaction mix was loaded onto a C₄ reverse phase-HPLC column (Vydac, Hisperia, Calif.) and products resolved isocratically at 32% acetonitrile. The β-endorphin peak was detected by absorbance at 214 nm using a Waters 484 detector. The reaction was quantitated by measuring the decrease in the β-endorphin peak area.

Example 7 Determination of Sites of Cleavage of Aβ Peptides

[0095] Purified insulysin was incubated with 25 μM Aβ₁₋₄₀ in 40 mM potassium phosphate buffer, pH 7.2, at 37° C. for 1 hour. The reaction products were loaded onto a C₄ reverse phase HPLC column and products resolved using a linear gradient of 5 to 75% acetonitrile over 65 minutes. Products were detected by absorbance at 214 nm using a Waters 484 detector and individual product peaks were collected manually. Product analysis was also conducted on an intact reaction mixture in which products were not resolved by HPLC. Products were identified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The reaction of insulysin with Aβ₁₋₄₂ was conducted in a similar manner with products identified by MALDI-TOF-MS directly from reaction mixtures.

Example 8 Aβ₁₋₄₀ Deposition Assay

[0096] Beta amyloid deposition assays were conducted as described by Esler et.al. (Esler et al. (1997) Nat Biotech 15:268-263). Briefly, 96 well microtiter plates pre-coated with aggregated amyloid β₁₋₄₀ (QCB/Biosource, Hopkinton, Mass.) were additionally coated with 200 μl of a 0.1% bovine serum albumin solution in 50 mM Tris-HCl, pH 7.5 for 20 minutes to prevent non-specific binding. For measuring Aβ₁₋₄₀ deposition in the presence or absence of insulysin, a 150 μl solution of 0.1 nM ¹²⁵I labeled Aβ₁₋₄₀ in 50 mM Tris-HCl, pH 7.5 was added to the pre-coated well and incubated for four hours. When added, insulysin (0.5 to 500 ng) was placed directly in the well at zero time. The reaction was stopped by washing off excess undeposited radiolabeled Aβ₁₋₄₀ with 50 mM Tris-HCl, pH 7.5. The radiolabel deposited onto the washed well was counted in a gamma counter. In a variation of this protocol, insulysin was preincubated with 1 nM ¹²⁵I-Aβ₁₋₄₀ for 60 minutes and then added to the deposition assay.

Example 9 Neuoroprotection Assays

[0097] Neurotoxicity assays were performed as described by Estus et.al. (Estus et al. (1997) J Neurosci 17:7736-7745) using embryonic day 18 rat fetuses to establish primary rat cortical neuron cultures. Rat brain cortical cells were initially cultured in AM₀ media for 3-5 hrs in 16 well chamber slides (Nalge Nunc International, Rochester, N.Y.) pre-coated with polyethyleneimine at a density of ˜1×10⁵ cells per well. The culture was enriched in neurons by replacement of the AM₀ media with Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Rockville, Md.) containing 100 units/ml penicillin, 100 μg/ml streptomycin and 2% B27 serum supplement (Life Technologies, Rockville, Md.).

[0098] Cells were treated with Aβ peptides and then fixed with 4% paraformaldehyde for 15 min. at room temperature. After washing the cells with PBS they were then stained with Hoechst 33258 at 1 μg/ml for 10 minutes. Neurons were then visualized by fluorescence microscopy. Those cells with uniformly dispersed chromatin were scored as survivors, while those cells containing condensed chromatin were scored as non-survivors. Readings were typically taken in triplicate with a minimum of 250 neurons scored from each well. Cells treated as described above were visualized using a Nikon microscope equipped with a Hoffman modulation contrast lens. Statistical analysis was performed on the samples using ANOVA.

Example 10 Immunofluorescence

[0099] The presence of aggregated Aβ₁₋₄₀ was detected in the neuronal cultures using the monoclonal antibody 10D5 (Walker et al. (1994) J Neuropathol Exp Neurol 53:377-383) at a 1:100 dilution in 5% goat serum in PBS. After an overnight incubation at 4° C. with this primary antibody, the wells were rinsed with PBS and incubated with a goat anti mouse secondary antibody conjugated to Cy-3 (Jackson ImmunoResearch, West Grove, Pa.) at a dilution of 1:250 in 5% goat serum in PBS. The wells were incubated at room temperature for 60 minutes and then after further washing with PBS, cells were examined under a fluorescence microscope.

Example 11 Results

[0100] To characterize the reaction of insulysin with the Aβ peptides, recombinant rat enzyme containing an amino-terminal His₆ affinity tag was expressed in baculovirus infected Sf9 cells. Expression of the enzyme in this system was high as evidenced by the ability to see insulysin protein in a crude extract by SDS-PAGE, FIG. 4. Purification of the recombinant enzyme was achieved by chromatography on a Ni-NTA-agarose column producing highly purified enzyme followed by chromatography on a Mono-Q column, which produced homogeneous enzyme, FIG. 4. The specific activity of the recombinant enzyme (2.6 μmols/min/mg) was comparable to enzyme purified from a thymoma cell line, EL-4 (3.3 μmols/min/mg), and thus the presence of the His₆ affinity tag had no discemable effect on enzyme activity.

[0101] To delineate the sites of cleavage of the Aβ₁₋₄₀ peptide by insulysin, the peptide was incubated with varying concentrations of the enzyme for one hour at 37° C., and then products were resolved by gradient reverse-phase HPLC. With 50 ng of insulysin, the lowest enzyme concentration used, three major cleavage sites at His¹⁴-Gln¹⁵ (peak 1), His¹³-His¹⁴ (peak 2), and Phe¹⁹-Phe²⁰ (peak 4 and peak 7) were discernable, TABLE 1 and FIG. 5. In addition, minor cleavage sites at Lys²⁸-Gly²⁹ (peak 5) and Phe²⁰-Ala²¹ (peak 6) was observed. When the amount of insulysin was increased to 250 ng, each of the products seen with 50 ng of enzyme increased, and an additional product corresponding to cleavage at Val¹⁸-Phe¹⁹ (peak 3) was observed. Further increasing insulysin to 500 ng showed a continued increase in each of the products. The same products were seen when Aβ₁₋₄₀ was treated with 500 ng of insulysin and analyzed by MALDI-TOF-MS without separation of the reaction products. It is interesting to note that one product peak Aβ₁₄₋₄₀ was not observed, while other product peaks were not apparent until after substantial metabolism had occurred. For example, Aβ₁₋₁₄ can be seen in the digest using 50 ng of insulysin while the product corresponding to the C-terminal half of this cleavage, Aβ₁₅₋₄₀, is not seen in the 50 ng reaction, but is observed with the 250 ng of enzyme. This is in part attributed to the hydrophobic nature of the C-terminal peptides and their greater retention times which produces HPLC peak broadening and decreased sensitivity. The overall cleavage profile is illustrated in FIG. 6.

[0102] The peaks from the HPLC chromatogram shown in FIG. 5 were collected and analyzed by MALDI-TOF. Product peaks are labeled sequentially in TABLE 1 as derived from HPLC (shown in FIG. 5). TABLE 1 Identification of products from insulysin cleavage of Aβ₁₋₄₀ Aβ₁₋₄₀ Peak no. Fragment Sequence 1  1-14 DAEFRHDSGYEVHH 2  1-13 DAEFRHDSGYEVH 3  1-18 DAEFRHDSGYEVHHQKLV 4  1-19 DAEFRHDSGYEVHHQKLVF 5  1-28 DAEFRHDSGYEVHHQKLVFFAEDVGSNK 6  1-20 DAEFRHDSGYEVHHQKLVFF 7 20-40 FAEDVGSNKGAIIGLMVGGVV 8 29-40 GAIIGLMVGGVV 9 21-40 AEDVGSNKGAIIGLMVGGVV 10 19-40 FFAEDVGSNKGAIIGLMVGGVV 11 15-40 QKLVFFAEDVGSNKGAIIGLMVGGVV

[0103] The Aβ₁₋₄₂ peptide was incubated with insulysin in an identical fashion as with Aβ₁₋₄₀ and the products were analyzed by MALDI-TOF mass spectrometry without prior separation by HPLC. Product peaks corresponding to cleavage at the His¹³-His¹⁴, His¹⁴-Gln ¹⁵, Phe¹⁹-Phe²⁰ and Phe²⁰-Ala²¹ positions were observed. These results indicate that both Aβ₁₋₄₀ and Aβ₁₋₄₂ are cleaved at the same sites. The rate of cleavage of 25 μM Aβ₁₋₄₀ was measured as 1.2 μmols/min/mg enzyme which indicates that the Aβ peptides are good substrates for insulysin.

[0104] The products of the action of insulysin on the Aβ peptides produces relatively large fragments. Since the peptide Aβ₂₅₋₃₅, which is derived from Aβ₁₋₄₀, is neurotoxic, it is possible that the products of insulysin action on the Aβ peptides could be toxic to neurons. To test this, rat cortical neurons were treated with Aβ peptides in the presence and absence of insulysin. Preliminary experiments were performed to obtain a suitable Aβ peptide concentration that would show a significant cytotoxic effect, as there are batch to batch variations in the ability of the Aβ peptides to mediate cytotoxic effects on cells in culture. These experiments established 30 μM Aβ₁₋₄₀ and 25 μM Aβ₁₋₄₂ as reasonable peptide concentrations which produce approximately 70% and 80% loss of cortical neurons respectively in 48 hrs.

[0105] The cell based assay using primary rat cortical neurons was used to determine whether the insulysin cleavage products of the Aβ peptides were themselves neurotoxic. Recombinant insulysin at concentrations ranging from 0.5 to 5000 ng was added simultaneously with the Aβ peptides to the cortical cultures. When added directly to the cultures as little as 50 ng of insulysin was effective in sparing the neurotoxic effects of Aβ₁₋₄₀ (FIG. 7A) while 500 ng of insulysin was effective in sparing the neurotoxic effects of Aβ₁₋₄₂ (FIG. 7B). This effect of insulysin is illustrated in FIG. 8 where cells were either stained with Hoechst 33258 to visualize DNA (panels A-D), with the Aβ antibody 10D5 to visualize cell associated Aβ (panels E-H), or visualized directly by Hoffman modulation microscopy (panels I-L). Using this phase contrast microscopy it can be seen that Aβ₁₋₄₀ caused the cells to appear shrunken (panel K) as compared to control cells which appear rounded (panel I). Aβ₁₋₄₀ induced chromatin condensation, which appears as small rounded nuclei (panel C), and Aβ cellular accumulation, which appears as a bright layering over the cells (panel G), is not evident in untreated cells (panels A & E). Cells to which insulysin was added along with Aβ₁₋₄₀ more closely resembled untreated cells (panels D, H and L). Also shown in FIG. 8 are controls in which cells were treated with insulysin alone (panels B, F and J).

[0106] During the progression of Alzheimer's disease monomeric Aβ peptides are deposited onto senile plaques. To test whether insulysin is able to prevent the deposition of the Aβ₁₋₄₀ peptide, a model system was used in which the deposition of radiolabeled Aβ₁₋₄₀ onto a synthetic amyloid plaque (synthaloid) is followed (Esler et al. (1999) Methods Enzymol 309:350-74). As seen in FIG. 9A, addition of insulysin at 0.5 ng to 500 ng with radiolabeled ¹²⁵I-Aβ₁₋₄₀ shows that 50 ng of insulysin is able to prevent the deposition of radiolabeled Aβ₁₋₄₀. FIG. 9B shows that preincubation of insulysin with radiolabeled ¹²⁵I-Aβ₁₋₄₀ for 60 minutes before adding it to the wells also shows that 50 ng insulysin is able to prevent the deposition of radiolabeled Aβ₁₋₄₀ onto the synthetic amyloid. We also conducted an experiment in which ¹²⁵I-Aβ₁₋₄₀ was first deposited onto the synthetic amyloid and then treated with insulysin to see if the enzyme could degrade pre-aggregated Aβ₁₋₄₀. After a 24 hr incubation with 5 μg of insulysin no radioactivity was released indicating that insulysin does not degrade aggregated Aβ peptides.

[0107] All of the references cited herein are incorporated by reference in their entirety.

[0108] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the following claims. 

What is claimed is:
 1. A method for preventing formation or growth of amyloid fibrils or plaques without causing neurotoxicity, comprising administering an inactivating effective amount of an amyloid peptide inactivating enzyme to a mammal in need thereof.
 2. The method according to claim 1, wherein said enzyme is a peptidase.
 3. The method according to claim 2, wherein said enzyme is insulin degrading enzyme (IDE) or neprilysin or endopeptidase 24.15, endopeptidase 24.16, or similar peptidases.
 4. The method according to claim 1, wherein said amyloid peptide is Aβ.
 5. The method according to claim 4, wherein said Aβ protein is Aβ1-40 or Aβ₁₋₄₂.
 6. The method according to claim 1, wherein said mammal is human.
 7. A method for preventing formation or growth of amyloid plaque without causing neurotoxicity, comprising: a) generating a recombinant viral or plasmid vector comprising a DNA sequence encoding an amyloid peptide inactivating enzyme operatively linked to a promoter; b) transfecting in vitro a population of cultured neural cells with said recombinant vector, resulting in a population of transfected neural cells; and c) transplanting said transfected neural cells by injection to the brain of a mammalian host, such that expression of said DNA sequence within said brain results in inactivation of said amyloid peptides.
 8. The method according to claim 7, wherein said neural cells are hippocampal or cortical cells.
 9. The method according to claim 7, wherein said enzyme is a peptidase.
 10. The method according to claim 9, wherein said peptidase is insulin degrading enzyme (IDE) or neprilysin.
 11. The method according to claim 7, wherein said amyloid peptide is Aβ.
 12. The method according to claim 11, wherein said Aβ protein is Aβ₁₋₄₀ or Aβ₁₋₄₂.
 13. The method according to claim 7, wherein said brain is the cortex or hippocampus.
 14. A method for preventing formation or growth of amyloid plaque without causing neurotoxicity, comprising: a) generating a recombinant viral or plasmid vector comprising a DNA sequence encoding an amyloid peptide inactivating enzyme operatively linked to a promoter; and b) injecting said vector to the brain of a mammalian host, such that expression of said DNA sequence within said brain results in inactivation of said amyloid peptides.
 15. A method for preventing formation or growth of amyloid plaque without causing neurotoxicity, comprising administering to a patient in need thereof a compound that enhances the expression of the amyloid inactivating enzyme.
 16. The method according to claim 15, wherein said enzyme is IDE or neprilysin.
 17. The method according to claim 15, wherein said enhancement of expression of the amyloid inactivating enzyme occurs at the gene expression level.
 18. The method according to claim 17, wherein said compound is a transactivator of said amyloid inactivating enzyme. 